This invention relates to catalytically active polypeptides and their use in making D-amino acids.
Unnatural or non-proteinogenic amino acids, which are structural analogs of the naturally-occurring amino acids that are the constituents of proteins, have important applications as pharmaceutical intermediates. For example, the anti-hypertensives ramipril, enalapril, benazapril, and prinivil are all based on L-homophenylalanine, and certain second generation pril analogs are synthesized from p-substituted-L homophenylalanine. Various β-lactam antibiotics use substituted D-phenylglycine side chains, and newer generation antibiotics are based on aminoadipic acid and other UAAs. The unnatural amino acids L-tert-leucine, L-nor-valine, L-nor-leucine, L-2-amino-5-[1,3]dioxolan-2yl-pentanoic acid, and the like have been used as a precursor in the synthesis of a number of different developmental drugs.
Unnatural amino acids are used almost exclusively as single stereoisomers. Since unnatural amino acids are not natural metabolites, traditional production methods for amino acids based on fermentation cannot generally be used since no metabolic pathways exist for their synthesis. Given the growing importance of unnatural amino acids as pharmaceutical intermediates, various methods have been developed for their enantiomerically pure preparation. Commonly employed methods include resolutions by diastereomeric crystallization, enzymatic resolution of derivatives, or separation by simulated moving bed (SMB) chiral chromatography. These methods can be used to separate racemic mixtures, but the maximum theoretical yield is only 50%.
In the case of non-proteinogenic L-amino acids such as L-nor-valine, L-nor-leucine, L-2-amino-5-[1-3]dioxolan-2-yl-pentanoic acid, L-tert-leucine, and many others, enzyme-catalyzed reductive amination is an effective method for their synthesis. Whereas the naturally-occurring alkyl and branched-chain amino acids can be produced by fermentation, taking advantage of the existing metabolic pathways to produce these amino acids, stereoselective production of non-proteinogenic analogs and various similar compounds is more difficult. The enzyme leucine dehydrogenase and mutants thereof have been shown to be capable of catalyzing the reductive amination of the corresponding 2-ketoacids of alkyl and branched-chain amino acids, and L-tert-leucine has been produced commercially with such an enzyme. A number of different reductive aminases for producing L-amino acids are commercially available currently (Enzyme catalog from BioCatalytics, Inc., Pasadena, Calif., March 2005).
However, to produce D-amino acids, enzyme-catalyzed reductive amination has not been an option in the past because enzymes catalyzing reductive amination of 2-ketoacids to produce D-amino acids have not been available. Accordingly, there is a need for novel mutant enzymes that catalyze the efficient reductive amination of a broad range of different 2-ketoacids to produce the corresponding D-amino acids, including D-counterparts of naturally-occurring amino acids and D-analogs of non-proteinogenic amino acids such as those listed above (D-nor-valine, D-nor-leucine, D-2-aminooctanoic acid, D-2-amino-5-[1,3]dioxolan-2yl-pentanoic acid, D-cyclohexylalanine, D-tert-leucine, and many others). There is also a need for new methods of making D-amino acids, using such mutant, D-amino acid dehydrogenase enzymes.
Optically pure D-amino acids are becoming increasingly important as pharmaceutically active compounds, chiral directing auxiliaries, and chiral synthons in organic synthesis. The largest current use of D-amino acids is in the production of semi-synthetic antibiotics. Ampicillin and amoxicillin (FIG. 1), made from D-phenylglycine and p-hydroxy-D-phenylglycine, respectively, are more broad-based and more stable to enzymatic degradation than naturally occurring (benzyl) penicillin. p-Hydroxy-D-phenylglycine is produced in a scale of several kilotons per year.
In addition to β-lactam antibiotics, D-amino acids are also found in antibacterial peptides. Table 1 lists several of these peptides along with the D-amino acids they contain.
TABLE 1Antibacterial peptides and the D-amino acids they contain.Antibacterial peptideD-Amino acidActinomycin DD-ValBacitracin AD-Glu, D-Phe, D-Orn, D-AspCirculinD-LeuGramicidin SD-PheFungisporinD-Phe, D-ValMalformin B1aD-Leu, D-CysMycobacillinD-AspPolymyxin B1D-PheTyrocidine AD-PheValinomycinD-Val
The naturally occurring amphibian skin peptides dermorphin and dermenkephalin are another class of bioactive peptides containing D-amino acids (D-alanine and D-methionine, respectively). These peptides are highly potent morphine-like agonist.
D-Cyclohexylalanine, a component of the drug Inogatran (AstraZeneca), is another example of a D-amino acid found in a pharmaceutical product. Inogatran (FIG. 2) is a direct, low molecular weight thrombin inhibitor used to prevent blood-clot formation and, when coupled with other drugs, used to stimulate thrombolysis.
Cetrorelix (Degussa) is also a currently produced D-amino acid-containing drug, prescribed to those with fertility problems. This drug blocks the effects of Gonadotropin Releasing Hormone and prevents premature ovulation. Premature ovulation can lead to eggs that are not ready for fertilization. Cetrorelix is also being investigated (currently in clinical test phase II) for the treatment of endometriosis and uterine fibroids in women, and benign prostatic hypertrophy in men. This drug is a decapeptide containing five D-amino acids (FIG. 3).
Another peptide containing all D-amino acids is currently being investigated for the treatment of HIV. This peptide is able to bind to an HIV coat protein known as gp41. Once the peptide is bound to gp41, the HIV virus is unable to fuse to human cells, and thus the peptide limits the spread of the HIV virus within the body. This is an example of retro-reverso peptide, where target peptides are made using reversed sequences of only D-amino acids. Many highly bioactive, stable peptide analogs have been produced this way, including antibacterials, HIV fusion protein inhibitors, and synthetic vaccines.
D-Amino acids are also used in the fine chemical industry. For example, the pyrethroid insecticide Fluvalinate contains D-valine as a key building block (FIG. 4). Fluvalinate is a synthetic pyrethroid which is used as a broad spectrum insecticide against moths, beetles, and other insect pests on a variety of plants including cotton, cereal, grape, potato, fruit tree, vegetable and plantation crops, fleas, and turf and ornamental insects. It has both stomach and contact activity in target insects.
Current Methods:
There are currently three predominant methods to produce D-amino acids—enzymatic resolution of the racemate, enzymatic synthesis using a D-amino acid transaminase, and the conversion of hydantoins using the coupled enzymatic reactions of a D-hydantoinase and D-carbamoylase. All of three methods have drawbacks, however.
In the enzymatic resolution method (FIG. 5) both enantiomers are chemically acylated at the amine or esterified at the carboxylate followed by enzymatic hydrolysis of only one stereoisomer using either an amidase or esterase (e.g. lipase). After enantioselective hydrolysis, the two compounds can be separated to give the optically pure L- or D-amino acid. This method is limited to a maximum theoretical yield of 50% per cycle; in actual practice the yield is typically between 30-40%. The resolution process also involves multiple sequential reaction and purification steps which cannot be done in a single pot. Also, in some cases it can be difficult to find an enzyme that can selectively hydrolyze only one of the two modified amino acids, limiting the breadth of scope of this method.
In the D-transaminase reaction (FIG. 6), a method developed at BioCatalytics, Inc., an amine is donated from a starting D-amino acid (donor) to a 2-ketoacid to form the D-amino acid of interest and the corresponding 2-ketoacid of the donor amino acid. This reaction is catalyzed by a D-amino acid transaminase. The donor amino acid must be of the D-form but, since L-amino acids are much cheaper (e.g. L-aspartate is ˜$3/kg), the transaminase reaction must be coupled with an amino acid racemase. This reaction can suffer from side reactions that generate byproducts, as the ketoacid formed from the donor amino acid can also be aminated. Special techniques are used to drive this reaction to completion. Typically L-aspartate is used as the donor and is converted to the D-antipode enzymatically; the 2-ketoacid formed, oxaloacetate, can spontaneously decarboxylate and drive the reaction (pyruvate is a poor amine acceptor). However, the decarboxylation step may not be sufficiently fast enough to keep up with the transaminase step, and yields and rates suffer in this case.
The D-hydantoinase/D-carbamoylase system (FIG. 7) starts with a racemate of hydantoins corresponding to the wanted amino acid. The conversion proceeds in two discrete steps. First, the D-hydantoin is selectively hydrolyzed to the D-carbamoylic acid with a D-hydantoinase, which is then hydrolyzed to the D-amino acid with a D-carbamoylase. The L-hydantoin will spontaneously racemize at a pH above 8, giving a theoretical yield of 100%. This method is currently used to make many D-amino acids; however, it does have some limitations. The conversion requires two types of enzymatic reactions that are difficult to carry out in a single pot, so two separate reactions and isolations are normally employed. This method is also dependent upon the spontaneous racemization of the hydantoin to achieve yields above 50%, and the rate of racemization is dependent on the substituent at the 5-position. This rate can vary greatly from τ1/2 of 0.3 h for phenylhydantoin (giving phenylalanine after hydrolysis), 5 h for benzylhydantoin (phenylglycine), 56 h for isopropylhydantoin (valine) and 120 h for tert-butylhydantoin (tert-leucine). Also, the substrate range of the D-hydantoinase and D-carbamoylase may not be broad enough to accept a wide range of substrate. Lastly, hydantoins are relatively insoluble in aqueous solution, limiting the titer of product that can be achieved.
Given the drawbacks of the three current methods for D-amino acid synthesis, there is clearly a need for a new method that reduces the number of steps necessary and increases the product yield.